We are interested in the molecular bases of the regulation of the actin-activated ATPase activities of class-I and class-II myosins, the biological roles of these myosins and the regulation of their biological activities. To understand the coupled contributions of myosin heads and tails to enzymatic activity and function, we are studying the properties of chimeras consisting of the motor domain of Dictyostelium myosin II and the tail domains of either Acanthamoeba or smooth muscle myosin II. The chimeras have 10-15 higher actin-activated ATPase activity than wild-type Dictyostelium myosin II but, in contrast to wild-type myosins, this activity is not regulated by phosphorylation of either the regulatory light chain or, in the Acanthamoeba chimera, phosphorylation of the tail. When expressed in myosin II-null cells, both chimeras rescue two essential functions of myosin II - cytokinesis in suspension culture and capping of con A receptors - but do not support full development to fruiting bodies. The structural requirements for localization of myosin II to the cleavage furrow of dividing Dictyostelium cells are under study. We have also deleted the hypertrophoic cardiomyopathy (HCM) surface loop of Dictyostelium myosin II or replaced it by the HCM loop of cardiac or smooth muscle myosin II, by the HCM loop of Acanthamoeba or Aspergillus myosin I, or modified the HCM loop by point mutations including the mutations of human cardiac myosin that cause the genetic disease hypertrophic cardiomyopathy. All of the HCM loop mutants rescue cytokinesis in suspension culture when expressed in Dictyostelium mysoin II-null mutants. None of the mutants appears to support full development, although these studies are not complete. We continue to study the structural basis of the regulation of the actin-activated ATPase activity of Acanthamoeba myosin II by phosphorylation of three sites in the tip of the tail by crystallography of appropriate tail constructs expressed in bacteria and by studying the enzymatic activity and regulation of heavy chain mutants.